How Do MicroRNAs Regulate Gene Expression?

Several thousand human genes, amounting to about one-third of the whole genome, are potential targets for regulation by the several hundred microRNAs (miRNAs) encoded in the genome. The regulation occurs posttranscriptionally and involves the ~21-nucleotide miRNA interacting with a target site in the mRNA that generally has imperfect complementarity to the miRNA. The target sites are almost invariably in the 3′-untranslated region of the messenger RNA (mRNA), often in multiple copies. Metazoan miRNAs were previously thought to down-regulate protein expression by inhibiting target mRNA translation at some stage after the translation initiation step, without much effect on mRNA abundance. However, recent studies have questioned these suppositions. With some targets, an increase in the rate of mRNA degradation by the normal decay pathway contributes to the decrease in protein expression. miRNAs can also inhibit translation initiation, specifically the function of the cap-binding initiation factor, eIF4E. Repressed target mRNAs as well as miRNAs themselves accumulate in cytoplasmic foci known as P-bodies, where many enzymes involved in mRNA degradation are concentrated. However, P-bodies may also serve as repositories for the temporary and reversible storage of untranslated mRNA, and reducing the expression (knockdown) of several distinct P-body protein components can alleviate miRNA-mediated repression of gene expression. The human genome encodes several hundred short (~21 residue) microRNAs (miRNAs), which are predicted to collectively regulate several thousand distinct genes, amounting to about one-third of all human genes. MicroRNAs generally interact with their target mRNAs through imperfect or incomplete complementary base-pairing to sites in the 3′-untranslated region of the messenger RNA (mRNA), which are usually present in multiple copies. These interactions result in a decrease in synthesis of the protein encoded by the mRNA, but until very recently, little was known about the mechanism(s) of this repression. An miRNA can decrease the intracellular concentration of its target mRNAs by accelerating the normal process of mRNA degradation and can inhibit the translation or decoding of the target mRNA, with the relative importance of these two mechanisms differing between different miRNA-mRNA pairs. The mechanism of inhibition of translation remains controversial, with some data pointing to inhibition of the translation initiation step, and other results indicative of inhibition at some later stage during the actual decoding of the mRNA sequence. This article reviews these recent data, discusses the controversies that remain unresolved, and makes suggestions for future research.

[1]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

[2]  P. Sarnow,et al.  Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific MicroRNA , 2005, Science.

[3]  Yang Yu,et al.  Evidence that microRNAs are associated with translating messenger RNAs in human cells , 2006, Nature Structural &Molecular Biology.

[4]  N. Standart,et al.  A conserved role of a DEAD box helicase in mRNA masking. , 2001, RNA.

[5]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[6]  C. Hellen,et al.  Translation Eukaryotic Initiation Factor 4G Recognizes a Specific Structural Element within the Internal Ribosome Entry Site of Encephalomyocarditis Virus RNA* , 1998, The Journal of Biological Chemistry.

[7]  Mihaela Zavolan,et al.  Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells , 2006, Nucleic acids research.

[8]  Ligang Wu,et al.  MicroRNAs direct rapid deadenylation of mRNA. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[9]  John G Doench,et al.  Recapitulation of short RNA-directed translational gene silencing in vitro. , 2006, Molecular cell.

[10]  D. Barford,et al.  Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity , 2004, The EMBO journal.

[11]  T. Rana,et al.  Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54 , 2006, PLoS biology.

[12]  D. Ballinger,et al.  The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates , 1983, Cell.

[13]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[14]  A. Nakamura,et al.  Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis. , 2004, Developmental cell.

[15]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[16]  D. Weil,et al.  The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules , 2005, Journal of Cell Science.

[17]  C. Smibert,et al.  Drosophila Cup is an eIF4E‐binding protein that functions in Smaug‐mediated translational repression , 2004, The EMBO journal.

[18]  Gregory J. Hannon,et al.  MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies , 2005, Nature Cell Biology.

[19]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.

[20]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[21]  N. Sonenberg,et al.  A newly identified N‐terminal amino acid sequence of human eIF4G binds poly(A)‐binding protein and functions in poly(A)‐dependent translation , 1998, The EMBO journal.

[22]  M. Mathews,et al.  Alterations of transcription and translation in HeLa cells exposed to amino acid analogs , 1984, Molecular and cellular biology.

[23]  J. Yates,et al.  A role for the P-body component GW182 in microRNA function , 2005, Nature Cell Biology.

[24]  R. Heintzmann,et al.  A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. , 2005, RNA.

[25]  A. Nakamura,et al.  Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. , 2001, Development.

[26]  W. Filipowicz,et al.  Inhibition of Translational Initiation by Let-7 MicroRNA in Human Cells , 2005, Science.

[27]  M. Hentze,et al.  Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes , 1997, Cell.

[28]  G. Hannon,et al.  Control of translation and mRNA degradation by miRNAs and siRNAs. , 2006, Genes & development.

[29]  David I. K. Martin,et al.  MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Hellen,et al.  Physical Association of Eukaryotic Initiation Factor 4G (eIF4G) with eIF4A Strongly Enhances Binding of eIF4G to the Internal Ribosomal Entry Site of Encephalomyocarditis Virus and Is Required for Internal Initiation of Translation , 2000, Molecular and Cellular Biology.

[31]  W. Henzel,et al.  Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz , 2003, The Journal of cell biology.

[32]  E. Miska,et al.  MicroRNA functions in animal development and human disease , 2005, Development.

[33]  Jerry Pelletier,et al.  Short RNAs repress translation after initiation in mammalian cells. , 2006, Molecular cell.

[34]  W. Filipowicz,et al.  Relief of microRNA-Mediated Translational Repression in Human Cells Subjected to Stress , 2006, Cell.

[35]  Roy Parker,et al.  Decapping and Decay of Messenger RNA Occur in Cytoplasmic Processing Bodies , 2003 .

[36]  Randal J. Kaufman,et al.  Stress granules and processing bodies are dynamically linked sites of mRNP remodeling , 2005, The Journal of cell biology.

[37]  A. Gingras,et al.  Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function , 1994, Nature.

[38]  Henning Urlaub,et al.  Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi , 2002, Cell.

[39]  R. Dooling,et al.  MASKING , 2008, The SAGE Encyclopedia of Research Design.

[40]  C. Hellen,et al.  Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry , 1996, Molecular and cellular biology.

[41]  W. Marzluff,et al.  The Histone 3′-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3 , 2002, Molecular and Cellular Biology.

[42]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[43]  Isabelle Behm-Ansmant,et al.  A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. , 2005, RNA.

[44]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[45]  Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. , 2001, Development.

[46]  E. Moss,et al.  Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. , 2002, Developmental biology.

[47]  E. R. Gavis,et al.  Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism , 2000, Current Biology.

[48]  Gary Ruvkun,et al.  Identification of many microRNAs that copurify with polyribosomes in mammalian neurons , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  R. Parker,et al.  Processing bodies require RNA for assembly and contain nontranslating mRNAs. , 2005, RNA.

[50]  N. Gray,et al.  The stem-loop binding protein stimulates histone translation at an early step in the initiation pathway. , 2005, RNA.

[51]  J. Castle,et al.  Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs , 2005, Nature.

[52]  Roy Parker,et al.  General Translational Repression by Activators of mRNA Decapping , 2005, Cell.

[53]  J. Richter,et al.  Human let-7a miRNA blocks protein production on actively translating polyribosomes , 2006, Nature Structural &Molecular Biology.

[54]  N. Sonenberg,et al.  A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay , 2005, The Journal of cell biology.

[55]  P. Bork,et al.  mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. , 2006, Genes & development.

[56]  A. Hatzigeorgiou,et al.  A combined computational-experimental approach predicts human microRNA targets. , 2004, Genes & development.

[57]  P. Sarnow,et al.  Initiation of Protein Synthesis from the A Site of the Ribosome , 2000, Cell.

[58]  R. Russell,et al.  Principles of MicroRNA–Target Recognition , 2005, PLoS biology.

[59]  A. Pasquinelli,et al.  Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation , 2005, Cell.

[60]  M. Hentze,et al.  Bruno Acts as a Dual Repressor of oskar Translation, Promoting mRNA Oligomerization and Formation of Silencing Particles , 2006, Cell.

[61]  Shuang Huang,et al.  Involvement of MicroRNA in AU-Rich Element-Mediated mRNA Instability , 2005, Cell.

[62]  S. Paulous,et al.  Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation , 2001, Molecular and Cellular Biology.

[63]  N. Standart,et al.  The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer. , 2004, Nucleic acids research.

[64]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[65]  Thomas Tuschl,et al.  RISC is a 5' phosphomonoester-producing RNA endonuclease. , 2004, Genes & development.

[66]  T. Tuschl,et al.  Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. , 2004, Molecular cell.

[67]  G. Merrill,et al.  Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[68]  W. Filipowicz,et al.  Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. , 2004, RNA.

[69]  S. Cohen,et al.  Genome-Wide Analysis of mRNAs Regulated by Drosha and Argonaute Proteins in Drosophila melanogaster , 2006, Molecular and Cellular Biology.

[70]  Yi Wen Kong,et al.  How do microRNAs regulate gene expression? , 2008, Biochemical Society transactions.

[71]  Artemis G Hatzigeorgiou,et al.  miRNP:mRNA association in polyribosomes in a human neuronal cell line. , 2004, RNA.

[72]  B. Séraphin,et al.  Cytoplasmic foci are sites of mRNA decay in human cells , 2004, The Journal of cell biology.

[73]  R. Jackson,et al.  The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes. , 2002, RNA.

[74]  Anton J. Enright,et al.  Zebrafish MiR-430 Promotes Deadenylation and Clearance of Maternal mRNAs , 2006, Science.

[75]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[76]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[77]  Roy Parker,et al.  Movement of Eukaryotic mRNAs Between Polysomes and Cytoplasmic Processing Bodies , 2005, Science.

[78]  B. Séraphin,et al.  The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies. , 2003, RNA.

[79]  Robert B. Russell,et al.  Principles of MicroRNATarget Recognition , 2005 .

[80]  Oliver Hobert,et al.  Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions , 2006, Nature Structural &Molecular Biology.

[81]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[82]  H. Blau,et al.  Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies , 2005, Nature Cell Biology.

[83]  M. Hentze,et al.  Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. , 2000, RNA.

[84]  Phillip D Zamore,et al.  Perspective: machines for RNAi. , 2005, Genes & development.

[85]  R. Jackson,et al.  A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. , 1998, Genes & development.